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PCR-RFLP Genotyping of Point Mutations in Caenorhabditis elegans
线虫点突变的PCR-RFLP基因型鉴定   

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Abstract

This protocol describes the basic principle of PCR/restriction digest genotyping of point mutations in worms, based on the principle of Restriction Fragment Length Polymorphism (RFLP) analysis. This type of genotyping is, particularly, useful when phenotypic analysis of animals carrying point mutations is difficult (e.g., in a complex genetic background).
I will illustrate the general procedures, using an example of daf-2 gene, encoding the sole insulin/IGF-1 receptor of C. elegans. Gems et al.(1998) did a very elegant job and characterized a series of mutations of daf-2, including the following two temperature-sensitive hypomorphic alleles:
daf-2(e1370): Substitution C/T (wild type/mutant), amino acid change: Missense P to S
Flanking sequences:
5’-CTCTATGAAATGGTTACACTCGGTGCTCAGCATATATTGGTTTGAGTAATGAGGTGT
Intracellular kinase domain, Class I, strong phenotype.
daf-2(e1368): Substitution C/T (wild type/mutant), amino acid change: Missense S to L
Flanking sequences:
5’-TCCGGAATTTACGTATTGAGGCAAAGTACTGTTCAGAAATVTATATGCTATCACAGT
Extracellular ligand binding domain, Class II, weak phenotype.
Here I will show you how to design the primers for PCR-RFLP analysis.
daf-2(e1370): Designed by Seung-Jae Lee from the Kenyon lab
Forward primer: 5’-CGGGATGAGACTGTCAAGATTGGAGATTTCGG-3’
Reverse primer: 5’-CAACACCTCATCATTACTCAAACCAATCCATG-3’
On the (-) strand, the nucleotide next to the 3’ end of reverse primer is G in wild-type allele, which is mutated to T in daf-2(e1370). Thus, by introducing another mutation (double C here, highlighted) into the reverse primer, it creates an Nco I-restriction site (i.e., CCATGG) only for PCR products derived from wild-type but NOT daf-2(1370).
daf-2(e1368): Designed by Peichuan Zhang from the Kenyon lab
Forward primer: 5’-GTTCCGGAATTTACGACGTATTGAGGCAACG-3’
Reverse primer: 5’-CTATCGGATCGAGTGGTATATTTAAC-3’
Similarly, on the (+) strand, the nucleotides next to the 3’ end of forward primer are TC in wild-type allele, and TT in daf-2(e1368). Thus, by introducing another mutation (C here, highlighted) into the forward primer, a restriction site of Acl I (i.e., AACGTT) is generated in the presence of daf-2(1368) point mutation.
The key is to introduce new mutation(s) at the 3’ end of one of your primers. Since the difference of the sizes of digest products is just ~30-bp, the length of the primer, you have to pick the other primer to generate an amplicon of ~200-bp – 250-bp or so.
Here is a website that can help you design the primers with appropriate restriction site for genotyping: http://helix.wustl.edu/dcaps/dcaps.html (dCAPS Finder 2.0) (Neff et al., 2002).

Keywords: C. elegans(线虫), PCR genotyping(PCR基因分型), Point mutation(点突变)

Materials and Reagents

  1. PK lysis buffer
  2. Proteinase K (Sigma-Aldrich, catalog number: P6556 )
  3. Common PCR reagent (e.g., Invitrogen PCR kit – Life Technologies, Invitrogen™, catalog number: 10342-020 ; or home-made Taq and buffer)
  4. Restriction enzymes (NEB)
  5. Agarose gel
  6. Ethidium bromide (Life Technologies, Invitrogen™, catalog number: 15585-011 )
  7. Plus DNA Ladder (Life Technologies, Invitrogen™, catalog number: 10787-018 )

Equipment

  1. MJ Research PTC-200 Thermo Cycler (MJ Research)
  2. Thin-wall PCR tubes (USA Scientific, catalog numbers: 1402-2700 or 1405-8100 )

Procedure

  1. Isolate genomic DNA with proteinase K digest.
    Tip 1. Typically, a large amount of PK lysis buffer is prepared (for the recipe, please refer to Caenorhabditis elegans/DNA/Single worm PCR) with supplement of proteinase K, and then small aliquots are stored (e.g., 1 ml) at -20 °C. This robust enzyme works well at a range from 20 μg/ml to 100 μg/ml, and the key is to activate it at 60 °C for ~1 h and then kill it at 95 °C for ~15 min or so. You do not want to see proteinase K torture your Taq enzyme during the subsequent PCR reaction.
    Tip 2. I prefer to pick reasonable numbers (e.g, 10) of gravid adult animals and stick them into a PCR tube with PK lysis buffer (e.g, 20 μl). It does not hurt to use more than 1 worm per PCR reaction (genomic DNA from ~1/2 worm works just fine for most robust PCR genotyping). For PCR-RFLP, it’d be better to use more than 1 worm per reaction (e.g., 5 to 10). However, too much DNA template, in some cases, may inhibit your PCR reactions.
  2. Perform a standard PCR, 20 μl per reaction.
    1. Set up the PCR, by adding the following component into a thin-wall PCR tube on ice in this order:
      DNAase-free ddH2O
      11.0 μl
      dNTP mix (10 mM each)
      0.4 μl (final, 200 μM each)
      Forward primer (10 μM)
      0.4 μl (final, 0.2 μM each)
      Reverse primer (10 μM)
      0.4 μl (final, 0.2 μM each)
      PCR buffer (10x)
      2.0 μl (final, 1x)
      MgCl2 (50 mM)
      0.8 μl (final, 2.0 mM)
      Worm lysates (20 μl)
      2.0 μl
      Taq (5 U/μl)
      0.1 μl (final, 0.5 U per reaction)
    2. Run PCR (put the tube on the block when it is hot):
      1 cycle
      94 °C, 3 min
      30 cycles
      94 °C, 10 sec; 58 °C, 30 sec; 72 °C, 30 sec
      1 cycle   
      72 °C, 10 min

  3. Digest with respective enzymes, 37 °C, O/N. Prepare multiplex (N+2) for N reactions:
    ddH2O
    2.5 μl
    10x buffer
    2.5 μl
    Enzyme (5 U/μl to 20 U/μl)
    0.2 μl
    Aliquot 5.0 μl into each PCR tube.

  4. Resolve O/N digest of PCR products on 2.0%-2.5% agarose gel.
    For daf-2(1370):
    Bands expected from Nco I-digest (NEB buffer 3): Wild-type, 202-bp; mutation, 234-bp.
    For daf-2(1368):
    Bands expected from Acl I-digest (NEB buffer 4 + BSA): Wild-type, 215-bp; mutation, 186-bp.
    Tip 3. Due to the small difference between the sizes of products, it is recommended to run the gel for a long time. Always add wild-type and mutants with known genotypes as controls. Ethidium bromide migrates toward cathode (-), just the opposite to the direction of DNA migration. To help subsequent visualization of DNA under UV light, it would be advised to add a few microliter of ethidium bromide (10 mg/ml) into the electrophoresis buffer near the anode (+).

Representative data



Figure 1. Representative data of PCR genotyping are shown here.
20 μl of daf-2(e1370) allele-genotyping PCR products were digested with Nco I at 37 ºC overnight. The digested DNA fragments were resolved on a 2.0% agarose gel. Expected sizes of DNA bands: wild-type, 202-bp; daf-2(e1370) mutation, 234-bp. Lane 1: 1 Kb Plus DNA Ladder. Lane 2, 7, 8: daf-2(e1370)+/+; Lane 4, 5: daf-2(e1370)+/-; Lane 3, 6: daf-2(e1370)-/-. The results are highly reproducible, and necessary controls should always be included to assure the results.

Recipes

  1. PK lysis buffer
    10 mM Tris-HCl (pH 8.0)
    50 mM KCl
    2.5 mM MgCl2
    0.45% Tween-20
    0.05% gelatin
    20 g/ml proteinase K
    Prepare and store small aliquots at -20 °C

Acknowledgments

This protocol was adapted from work performed by members of the Kenyon lab, including PZ. PZ was supported by a postdoctoral fellowship from the Larry Hillblom Foundation.

References

  1. Gems, D., Sutton, A. J., Sundermeyer, M. L., Albert, P. S., King, K. V., Edgley, M. L., Larsen, P. L. and Riddle, D. L. (1998). Two pleiotropic classes of daf-2 mutation affect larval arrest, adult behavior, reproduction and longevity in Caenorhabditis elegans. Genetics 150(1): 129-155.
  2. Neff, M. M., Turk, E. and Kalishman, M. (2002). Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 18(12): 613-615.

简介

该协议描述了基于限制性片段长度多态性(RFLP)分析的原理的蠕虫中点突变的PCR /限制性消化基因分型的基本原理。当具有点突变的动物的表型分析困难时(例如在复杂遗传背景中),这种类型的基因分型特别有用。
我将用一个编码C的唯一胰岛素/IGF-1受体的daf-2基因的实例来说明一般程序。 elegans 。 Gems等人(1998)做了非常优雅的工作,并且表征了daf-2的一系列突变,包括以下两个温度敏感的hypomorphic等位基因:
daf-2(e1370):取代C/T(野生型/突变体),氨基酸改变:错配P至S
侧翼序列:
5'-CTCTATGAAATGGTTACACTCGGTGCTCAGCATATATTGGTTTGAGTAATGAGGTGT < br />细胞内激酶结构域,I类,强表型。
daf-2(e1368):替换C/T(野生型/突变体),氨基酸改变:错误S到L
侧翼序列:
5' -TCCGGAATTTACGTATTGAGGCAAAGTACTGTTCAGAAATVTATATGCTATCACAGT
细胞外配体结合域II类,弱表型。
在这里,我将向您展示如何设计用于PCR-RFLP分析的引物。
daf-2(e1370):由Ken-on实验室Seung-Jae Lee设计
正向引物:5'-CGGGATGAGACTGTCAAGATTGGAGATTTCGG-3'
反向引物:5 '-CAACACCTCATCATTACTCAAACCAATCCATG-3'
在( - )链上,反向引物的3'末端旁边的核苷酸是野生型等位基因中的G,其在daf-2中突变为T(e1370 )。因此,通过在反向引物中引入另一突变(在此为双C,突出显示),其仅对来源于野生型的PCR产物产生Nco I限制性位点(即 CCATGG) em> daf-2(1370)。
daf-2(e1368):由Pexy Zh??ang从Kenyon实验室设计
正向引物:5'-GTTCCGGAATTTACGACGTATTGAGGCAACG-3'
反向引物: CTATCGGATCGAGTGGTATATTTAAC-3'
类似地,在(+)链上,正向引物的3'末端旁边的核苷酸在野生型等位基因中是TC,在daf-2(e1368) em>。因此,通过在正向引物中引入另一个突变(C,突出显示),在daf-2(1368)存在下产生Ac11(,AACGTT)的限制性位点, 点突变。
关键是在你的一个引物的3'端引入新的突变。因为消化产物大小的差异只是约30-bp,引物的长度,你必须选择另一个引物产生?200-bp-250-bp左右的扩增子。
这里是一个网站,可以帮助您设计具有合适的限制性位点基因分型的引物:http://helix.wustl.edu/dcaps/dcaps.html(dCAPS Finder 2.0)(Neff et al。 ,2002)。

关键字:线虫, PCR基因分型, 点突变

材料和试剂

  1. PK裂解缓冲液
  2. 蛋白酶K(Sigma-Aldrich,目录号:P6556)
  3. 常用PCR试剂(例如Invitrogen PCR试剂盒 - Life Technologies,Invitrogen TM,目录号:10342-020;或自制Taq和缓冲液)
  4. 限制酶(NEB)
  5. 琼脂糖凝胶
  6. 溴化乙锭(Life Technologies,Invitrogen TM,目录号:15585-011)
  7. Plus DNA Ladder(Life Technologies,Invitrogen TM,目录号:10787-018)

设备

  1. MJ Research PTC-200热循环仪(MJ Research)
  2. 薄壁PCR管(USA Scientific,目录号:1402-2700或1405-8100)

程序

  1. 用蛋白酶K消化分离基因组DNA 提示1。通常,制备大量的PK裂解缓冲液(对于配方,请参考秀丽隐杆线虫/DNA /单蠕虫PCR),补充蛋白酶K ,然后在-20℃下储存小等分试样(例如,1ml)。该强效酶在20μg/ml至100μg/ml的范围内工作良好,关键是在60℃下活化约1小时,然后在95℃下将其杀死约15分钟左右。你不想看到蛋白酶K在随后的PCR反应过程中折磨你的Taq酶 提示2。我更喜欢选择合理的数字(例如,10)的妊娠成年动物,并将它们放入装有PK裂解缓冲液的PCR管中20μl)。它不伤害每个PCR反应使用超过1个蠕虫(〜1/2蠕虫的基因组DNA工作正好适合大多数强大的PCR基因分型)。对于PCR-RFLP,最好每个反应使用超过1个蠕虫(例如,5至10)。但是,在某些情况下,太多的DNA模板可能会抑制PCR反应
  2. 执行标准PCR,每个反应20μl。
    1. 设置PCR,通过在冰上按以下顺序添加以下组分到薄壁PCR管中:
      无DNA酶的ddH 2 O 2/b 11.0微升
      dNTP混合物(各10mM) 0.4μl(最终,每种200μM)
      正向引物(10μM)
      0.4μl(最终,每种0.2μM)
      反向引物(10μM)
      0.4μl(最终,每种0.2μM)
      PCR缓冲液(10x)
      2.0μl(最终,1x)
      MgCl 2(50mM)
      0.8μl(终浓度,2.0mM)
      蠕虫裂解物(20μl)
      2.0μl
      Taq(5U /μl)
      0.1μl(最终,每次反应0.5U)
    2. 运行PCR(当热块时将管放在块上):
      1个周期
      94℃,3分钟
      30个周期
      94℃,10秒; 58℃,30秒; 72℃,30秒
      1周   
      72℃,10分钟

  3. 用相应的酶消化,37℃,O/N。 准备多重(N + 2)N反应:
    ddH sub 2 O
    2.5μl
    10x缓冲区
    2.5μl
    酶(5U /μl至20U /μl)
    0.2μl
    等分5.0μl到每个PCR管中
  4. 在2.0%-2.5%琼脂糖凝胶上分析PCR产物的O/N消化 对于 daf-2(1370):
    预期来自Nco I-消化物(NEB缓冲液3)的条带:野生型,202-bp;突变,234 bp 对于 daf-2(1368):
    预期来自Acl 1-消化物(NEB缓冲液4 + BSA)的条带:野生型,215-bp;突变,186 bp 提示3。由于产品尺寸之间的微小差异,建议长时间运行凝胶。总是添加具有已知基因型的野生型和突变体作为对照。溴化乙锭向阴极( - )迁移,正好与DNA迁移的方向相反。为了帮助随后在UV光下观察DNA,建议在阳极(+)附近的电泳缓冲液中加入几微升溴化乙锭(10mg/ml)。

代表数据



图1.这里显示了PCR基因分型的代表性数据。20μldaf-2(e1370)等位基因 - 基因分型PCR产物用Nco 我在37℃过夜。消化的DNA片段 在2.0%琼脂糖凝胶上分离。 DNA条带的预期大小:野生型,202-bp; daf-2(e1370)突变,234-bp。 泳道1:1Kb Plus DNA梯。 泳道2,7,8:daf-2(e1370) +/+ ; 泳道4,5:daf-2(e1370) +/- ; 泳道3,6:daf-2(e1370) -/- 。 结果是高度可重复的,并且必须包括必要的控制以确保结果。

食谱

  1. PK裂解缓冲液
    10mM Tris-HCl(pH8.0) 50 mM KCl
    2.5mM MgCl 2 v/v 0.45%Tween-20
    0.05%明胶
    20μg/ml蛋白酶K
    准备并在-20°C下保存小份。

致谢

该协议改编自Kenyon实验室的成员(包括PZ)所进行的工作。 PZ由Larry Hillblom基金会的博士后研究金支持。

参考文献

  1. Gems,D.,Sutton,A.J.,Sundermeyer,M.L.,Albert,P.S.,King,K.V.,Edgley,M.L.,Larsen,P.L.and Riddle,D.L。(1998)。 两种多效性的daf-2突变类型影响幼虫的捕获,成人行为,生殖和长寿 > Caenorhabditis elegans 。 150(1):129-155。
  2. Neff,M.M.,Turk,E。和Kalishman,M。(2002)。 用于单核苷酸多态性分析的基于网络的引物设计。 趋势基因 18(12):613-615。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, P. (2012). PCR-RFLP Genotyping of Point Mutations in Caenorhabditis elegans. Bio-protocol 2(6): e128. DOI: 10.21769/BioProtoc.128.
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Hi Peichuan,

Nice protocol!

I have a few questions about the electrophoresis condition you use: Do you use regular agarose for the gel? And do you use TAE or TBE buffer? I'm deciding whether to use agarose gel or acrylamide gel, and can you give me some suggestions?

Thank you very much!

Sophia
2/17/2012 2:09:32 AM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Sophia,

Thanks! I'm glad that our protocols have attracted attention from potential users.

We have been using regular agarose gel (2.0%) and 1X TAE buffer all the time. In my case, I need to separate two bands of about 200-bp (with difference of ~30-bp), and sometimes, I used 2.5% agarose gel instead. Try to not use too high voltage to avoid "smiling effects", and always remember to have positive and negative controls for comparison. Also use fresh 1X TAE buffer, as its buffering capacity drops significantly after a few rounds. You may consider designing a few different PCR-restriction digest strategies and pick the one that produces best resolution.

I only used TBE buffer, which is better than TAE in its buffering capacity, for gel shifting experiments. I recall that I saw some notes about achieving better resolution with TBE for smaller DNA (<300-bp on 2% agarose gel).

Hope this would help you a bit. Please let me know if you have further questions, and best luck with your experiments.

Peichuan

2/17/2012 7:20:27 AM


Sophia
VT

Hi Peichuan,

Thank you so much for your detailed explanation! I'm going to try out your protocol and will let you know of my progress.

Sophia

2/17/2012 11:42:05 PM


Jimmy
Tufts University Medical School
Hi Peichuan,

I actually had some success using Taq w/out exo ability from NEB. I saw the restriction cut product band shift ~30 bp lower than the uncut product (as well as a 30BP band faintly below). So thank you for the suggestion! Since we have a DNA synthesis core here it was very easy and quick to get new primers.

Interestingly I continued 1 last effort to see if the phosphorothioate bond would at least HELP prevent exonuclease activity, so I performed touchdown PCR and added 3%DMSO and decreased dNTP (all to increase the specificity) and I actually got a product that was successfully cut by enzyme, but only half of the product. I assume this means the DNA I'm examining is heterozygous for the mutation (Diploid organism). My reasoning is that maybe with added specificity of TD PCR the Hi Fidelity enzyme is chewing back only on the non-complementary chromosomal DNA but leaving the complementary (except for the 1 pt mut) intact? And if so, does this mean non-proofreading taq cannot distinguish between homozygous and heterozygous?

Thank you very much, and sorry for accidentally posting 3 times last time! :)
8/19/2011 4:10:18 AM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Jimmy,

Very glad that it worked out well for you. And thanks so very much for sharing your own experience, which is great and would definitely help us improve the protocol as provided on our website. We hope to provide more high-quality author-validated protocols to share among our research community.

I believe that you have taken right action for PCR, by using modified primer, touchdown strategy and DMSO, to improve amplification specificity/efficacy in your case.

Most Taq enzymes, unless otherwise modified, do not possess 3'->5' proof-reading exonuclease activities. Please check the info that I have found from NEB
http://www.neb.com/nebecomm/products/faqproductm0273.asp

In my case of daf-2 genotyping, I could actually distinguish heterozygote from homozygote. I could tell about two bands for heterozygotes, with wild type and daf-2 homozygotes as controls. I also used just regular desalted primers for genotyping.

BTW, to confirm the PCR genotyping results, you can also use regular Taq to amplify a PCR product (e.g., 500-bp or so) around the mutation, and then sequence the product with a primer.

Best,
Peichuan

8/19/2011 7:22:19 AM


Jimmy
Tufts University Medical School
Hi,

I have actually being trying to do almost this exact same thing, only I have had no luck introducing a point mutant at the 3' ends of my primers in order to complete a restriction site (in the mutant version only). I believe its because I'm using a proofreading enzyme which sees the base mismatch and cleaves it off, so I ordered a primer with a phosphorothioate modification to prevent exonuclease activity by the DNA pol, but I am still having no luck. Should I switch to a non-proofreading enzyme (regular taq)? And when you perform your restriction digest do you see the 30BP band or do you just look for a decrease in your 200BP band?

Thank you very much!

Jimmy
8/17/2011 9:42:26 PM Reply
Peichuan Zhang
Department of Biology, The Pennsylvania State University, USA

Hi Jimmy,

I used very typical recombinant Taq (e.g., Invitrogen 10342-020) and it worked pretty well in my hand (home-made Taq also worked). I would recommend you not to use Taq w/ high proof-reading capacity.

The other thing that I have in mind is that you probably would like to re-design your primers. Put GC clamp at 5' end, but not more than 4 GC at the 3'-end. Treat them as primers for qPCR.

I run the gel for rather a long time (put a little bit of EtBr in the buffer), with the aim to separate the two bands, which differ just in ~30-bp or so (I don't really bother to check the 30-bp band though). In my case, I can always check the phenotypic readout for daf-2 mutations, which is dauer formation. If you have concern about the genotyping, you should also sequence the PCR product and check certain phenotype that are associated with the mutation.

Best luck,
Peichuan

8/18/2011 1:01:21 PM